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M Ehrenberg

Publications and source records attributed to M Ehrenberg.

At least 37 records · Page 2Linked to original sources
PubMed2000

Fusidic acid-resistant EF-G perturbs the accumulation of ppGpp.

Reductions in growth rate caused by fusidic acid-resistant EF-G mutants in Salmonella typhimurium correlate strongly with increased mean cell size. This is unusual because growth rate and cell size normally correlate positively. The global transcription regulator molecule ppGpp has a role in co-ordinating growth rate and division, and its basal level normally correlates inversely with cell size at division. We show that fusidic acid-resistant EF-G mutants have perturbed ppGpp basal levels during steady-state growth and perturbed induced levels during starvation. One mutation, fusA1, associated with the slowest growth rate and largest cell size, causes a reduction in the basal level of ppGpp to one-third of that found in the wild-type strain. Other fusA mutants with intermediate or wild-type growth rates and cell sizes have either normal or increased basal levels of ppGpp. There is an inverse relationship between the basal level of ppGpp in vivo and the degree to which translation dependent on mutant EF-G is inhibited by ppGpp in vitro. This enhanced interaction between mutant EF-G and ppGpp correlates with an increased KM for GTP. Our results suggest that mutant EF-G modulates the production of ppGpp by the RelA (PSI) pathway. In conclusion, fusidic acid-resistant EF-G mutations alter the level of ppGpp and break the normal relationship between growth rate and cell size at division. It would not be surprising if other phenotypes associated with these mutants, such as loss of virulence, were also related to perturbations in ppGpp levels effected through altered transcription patterns.

Anti-Bacterial Agents↗
PubMed1999

Activities of constitutive promoters in Escherichia coli.

The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+ spoT+ strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient DeltarelADeltaspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the beta-lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage lambda; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of a lac deletion strain. The amount of 5'-terminal lacZ mRNA and of beta-galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combining lacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+ spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity of rrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+ spoT+) relative to ppGpp-deficient (DeltarelA DeltaspoT) bacterial strains.

Cell Division↗
PubMed1999

Shutdown in protein synthesis due to the expression of mini-genes in bacteria.

Mutants of Escherichia coli partially deficient in peptidyl-tRNA hydrolase are killed by the expression of certain very short open reading frames (mini-genes), encoded by the wild-type bar regions of phage lambda. According to the current hypothesis, protein synthesis is shut off, and the host cells die, after essential tRNA species become sequestered due to abnormal translation termination (drop-off) of mini-gene-encoded peptides as peptidyl-tRNA. Here we study variants of bar mini-genes, both in vivo and in vitro, in order to identify the structural elements that influence this inhibition of protein synthesis. Three parameters were measured during the expression of these variants: the rates of normal translation termination, peptidyl-tRNA dissociation from the ribosome and hydrolysis of peptidyl-tRNA by peptidyl-tRNA hydrolase were measured. Previous observations that RRF, EF-G and RF3 stimulated drop-off were confirmed and extended; stimulation by these factors can reach 30-fold. Both factor-stimulated and spontaneous drop-off depended on the nature of the stop signal. The degree of inhibition of cell growth following induction of mini-gene expression could be accounted for in terms of a toxicity index comprising the three parameters above. Inhibition was greatly reduced in cells lacking RF3. Mini-genes with more efficient Shine/Dalgarno sequences killed cells even with normal peptidyl-tRNA hydrolase activity. It is proposed that the retranslation by ribosomes of mini-gene transcripts with efficient ribosome binding (Shine/Dalgarno) sequences strongly contributes to the inhibitory effects of mini-gene expression on protein synthesis.

Bacterial Proteins↗
PubMed1999

Decay of rplN and lacZ mRNA in Escherichia coli.

To examine the previously proposed retroregulation model of spc mRNA degradation, two strains of Escherichia coli B/r were used; one has wild-type spc and lac operons and the other has a lac operon deletion, a wild-type spc operon, and a Pspc-rplN-lacZ fusion operon lacking the normal control sites of the spc operon (rplN is the first gene in the spc operon of ribosomal proteins). The decay of rplN mRNA and of lacZ mRNA in these strains was determined during exponential growth at different rates and after transcript initiation was inhibited by the antibiotic rifampicin. Functional decay of lacZ mRNA was monitored by measurements of beta-galactosidase activity and chemical decay was monitored using probes complementary to rplN, rplX, and to the 5' and 3'-terminal sections of lacZ. Analysis of the data was based on the assumption that the decay involves an endonucleolytic cleavage that functionally inactivates the mRNA and that this is followed by exonucleolytic degradation of the cleavage products. The major conclusions were: (1) During exponential growth, lacZ mRNA of the lac operon was translated about twice as frequently as lacZ mRNA of the spc-lac fusion, and both kinds of lacZ mRNA were translated at an elevated rate in the presence of rifampicin. (2) For lacZ mRNA from the lac operon, the endonuclease inactivation reaction was not affected by rifampicin, but the exonuclease reaction was inhibited. (3) The decay of rplN mRNA from the spc operon was accelerated in the presence of rifampicin; the average life was estimated to be six minutes during exponential growth in LB medium, and 2.8 minutes in the presence of rifampicin. (4) The decay of the rplN section of mRNA from the spc-lac operon fusion was coupled to the decay of the downstream lacZ mRNA section and was strongly inhibited (i.e. partially blocked) in the presence of rifampicin. These results show that the decay of spc mRNA differs in some important aspects from the decay of lac mRNA and support the retroregulation model. Moreover, the results indicate that rifampicin can have a significant and selective impact on the kinetics of both mRNA translation and decay.

Escherichia coli↗
PubMed1999

Initiation of Escherichia coli ribosomes on matrix coupled mRNAs studied by optical biosensor technique.

The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, has been used to study the initiation of protein synthesis by E. coli ribosomes on surface coupled mRNA. mRNA was first periodate oxidized and then hydrazide coupled to the surface of a CM5 sensor chip. The formation of initiation complexes on the surface coupled mRNA was monitored in real-time with a BIACORE 2000 instrument. Mature 70S*mRNA*fMet-tRNA(Met) initiation complexes were assembled on mRNA by sequential introduction of the 30S and 50S subunits supplemented with appropriate initiation factors and fMet-tRNA(Met). We show that the formation of 70S*mRNA complexes on the surface coupled mRNA proceeds efficiently only in the presence of tRNA. Moreover, 70S*mRNA*fMet-tRNA(Met) complexes formed with fMet-tRNA(Met) are more stable than similar complexes formed with deacylated tRNAs. The efficient formation and slow dissociation of mature 70S*mRNA*fMet-tRNA(Met) initiation complexes are most easily explained by the stabilization of the interaction of the ribosomal subunits by fMet-tRNA(Met). This work demonstrates the feasibility of the BIACORE technique for studying the initiation of protein synthesis.

Base Sequence↗
PubMed1999

Novel roles for classical factors at the interface between translation termination and initiation.

The pathway of bacterial ribosome recycling following translation termination has remained obscure. Here, we elucidate two essential steps and describe the roles played by the three translation factors EF-G, RRF, and IF3. Release factor RF3 is known to catalyze the dissociation of RF1 or RF2 from ribosomes after polypeptide release. We show that the next step is dissociation of 50S subunits from the 70S posttermination complex and that it is catalyzed by RRF and EF-G and requires GTP hydrolysis. Removal of deacylated tRNA from the resulting 30S:mRNA:tRNA posttermination complex is then necessary to permit rapid 30S subunit recycling. We show that this step requires initiation factor IF3, whose role was previously thought to be restricted to promoting specific 30S initiation complex formation from free 30S subunits.

Acetylation↗
PubMed1998

A direct estimation of the context effect on the efficiency of termination.

An in vitro assay in which terminating Escherichia coli ribosomes with different stop signals in the A-site compete for a limited amount of a release factor (RF1 or RF2) has been used to estimate the relative termination efficiencies at stop codons with different adjacent downstream nucleotides. The assay allows direct measurements of relative kcat/Km parameters for the productive association of release factors to ribosomes. The kcat/Km parameter is larger for UAA(U) than for UAA(C) programmed ribosomes and the difference in kcat/Km is much larger for RF2 (about 80%) than for RF1 (about 30%). These differences in the kcat/Km parameter are not affected by the addition of release factor RF3. The only discernible effect of RF3 is a considerable acceleration of RF1/2 recycling.The estimated kcat/Km parameters correlate well with the affinities of release factors for ribosomes programmed with different stop signals. These affinities were estimated from the extent of inhibition of ribosomal recycling by high concentrations of release factors in the absence of release factor RF3. The affinity for RF2 depends on the immediate downstream context of the stop codon in the translated mRNA and is about three times higher for UAA(U) than for UAA(C). The corresponding difference in affinities for RF1 is twofold. For all stop signals studied, the estimated affinity of RF2 for terminating ribosomes is much lower than that of RF1. It is also striking that the affinity of ribosomes for a chromosomally expressed RF2 is at least three times higher than for RF2 isolated from an overproducing E. coli strain.

Base Sequence↗
PubMed1998

Initiation factors IF1 and IF2 synergistically remove peptidyl-tRNAs with short polypeptides from the P-site of translating Escherichia coli ribosomes.

A novel function of initiation factors IF1 and IF2 in Escherichia coli translation has been identified. It is shown that these factors efficiently catalyse dissociation of peptidyl-tRNAs with polypeptides of different length from the P-site of E. coli ribosomes, and that the simultaneous presence of both factors is required for induction of drop-off. The factor-induced drop-off occurs with both sense and stop codons in the A-site and competes with peptide elongation or termination. The efficiency with which IF1 and IF2 catalyse drop-off decreases with increasing length of the nascent polypeptide, but is quite significant for hepta-peptidyl-tRNAs, the longest polypeptide chains studied. In the absence of IF1 and IF2 the rate of drop-off varies considerably for different peptidyl-tRNAs, and depends both on the length and sequence of the nascent peptide. Efficient factor-catalysed drop-off requires GTP but not GTP hydrolysis, as shown in experiments without guanine nucleotides, with GDP or with the non-cleavable analogue GMP-PNP.Simultaneous overexpression of IF1 and IF2 in vivo inhibits cell growth specifically in some peptidyl-tRNA hydrolase deficient mutants, suggesting that initiation factor-catalysed drop-off of peptidyl-tRNA can occur on a significant scale in the bacterial cell. Consequences for the bacterial physiology of this previously unknown function of IF1 and IF2 are discussed.

Carboxylic Ester Hydrolases↗
PubMed1998

Solution structure of the ternary complex between aminoacyl-tRNA, elongation factor Tu, and guanosine triphosphate.

Complex formation between elongation factor Tu (EF-Tu), Phe-tRNAPhe, and GTP was analyzed by small-angle neutron and X-ray scattering methods. Both techniques show that the ternary complex consists of one EF-Tu and one aminoacyl-tRNA. No shift in stoichiometry was detected when the temperature was raised from 5 to 37 degreesC, in contrast to previous observations obtained from RNase A protection experiments [Bilgin and Ehrenberg (1995) Biochemistry34, 715-719]. A small but significant increase in the radius of gyration of the complex was observed when the temperature was decreased from 37 to 5 degreesC. The X-ray solution scattering patterns were compared with those calculated from the crystal structure of the complex formed between EF-Tu from Thermus aquaticus and Phe-tRNAPhe from yeast. The comparison shows that the solution structure of the ternary complex, formed entirely from Escherichia coli components and under translationally optimal buffer conditions, is very close to the crystal structure, formed from heterologous components under very different conditions. Furthermore, for the hybrid complex in solution there is no evidence for the formation of trimers as suggested by the crystal structure.

Binding Sites↗
PubMed1998

Trade-off between segregational stability and metabolic burden: a mathematical model of plasmid ColE1 replication control.

A model of ColE1 copy number control has been developed where molecular details of replication are connected both to segregational stability and metabolic burden. Efficient replication control reduces copy number variation and increases segregational stability for a given average copy number. Copy number variation is predicted to depend on the type of inhibition mechanism as well as RNA I and RNA II turnover rate constants. It is shown that when both RNA I and RNA II transcription frequencies and the rate constant for degradation of free RNA I are very large, a hyperbolic inhibition mechanism must compensate with a 1.4 times greater average copy number to obtain the same segregational stability as an exponential inhibition mechanism. How sensitively the replication frequency responds to changes in RNA I concentration depends on the type of inhibition mechanism and the number of attempts to form an RNA II replication primer per plasmid and cell cycle. If RNA I is too stable, it will not follow changes in plasmid concentration closely, and when the transcription frequency for RNA I is only slightly higher than for RNA II, RNA I concentration becomes randomized. In both these cases, the proportionality between the single cell RNA I and plasmid concentrations is lost and this impairs copy number control. Thresholds in the rate for degradation of free RNA I as well as in RNA I and RNA II transcription frequencies have been computed, where an increase in these rate constants has a negligible effect on segregational stability but a corresponding decrease leads to segregational disaster. This indicates that there exists a well defined optimal set of rate constants where the regulation system works well without excessive metabolic load. A number of new experiments are suggested to address features of particular importance for the evolution of ColE1 copy number control.

DNA Replication↗
PubMed1998

Mutations in RNAs of both ribosomal subunits cause defects in translation termination.

Mutations in RNAs of both subunits of the Escherichia coli ribosome caused defects in catalysis of peptidyl-tRNA hydrolysis in a realistic in vitro termination system. Assaying the two codon-dependent cytoplasmic proteins that drive termination, RF1 and RF2, we observed large defects with RF2 but not with RF1, a result consistent with the in vivo properties of the mutants. Our study presents the first direct in vitro evidence demonstrating the involvement of RNAs from both the large and the small ribosomal subunits in catalysis of peptidyl-tRNA hydrolysis during termination of protein biosynthesis. The results and conclusions are of general significance since the rRNA nucleotides studied have been virtually universally conserved throughout evolution. Our findings suggest a novel role for rRNAs of both subunits as molecular transmitters of a signal for termination.

Base Sequence↗
PubMed1998

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.

Bacterial Proteins↗
PubMed1998

Requirements for rapid plasmid ColE1 copy number adjustments: a mathematical model of inhibition modes and RNA turnover rates.

The random distribution of ColE1 plasmids between the daughter cells at cell division introduces large copy number variations. Statistic variation associated with limited copy number in single cells also causes fluctuations to emerge spontaneously during the cell cycle. Efficient replication control out of steady state is therefore important to tame such stochastic effects of small numbers. In the present model, the dynamic features of copy number control are divided into two parts: first, how sharply the replication frequency per plasmid responds to changes in the concentration of the plasmid-coded inhibitor, RNA I, and second, how tightly RNA I and plasmid concentrations are coupled. Single (hyperbolic)- and multiple (exponential)-step inhibition mechanisms are compared out of steady state and it is shown how the response in replication frequency depends on the mode of inhibition. For both mechanisms, sensitivity of inhibition is "bought" at the expense of a rapid turnover of a replication preprimer, RNA II. Conventional, single-step, inhibition kinetics gives a sloppy replication control even at high RNA II turnover rates, whereas multiple-step inhibition has the potential of working with unlimited precision. When plasmid concentration changes rapidly, RNA I must be degraded rapidly to be "up to date" with the change. Adjustment to steady state is drastically impaired when the turnover rate constants of RNA I decrease below certain thresholds, but is basically unaffected for a corresponding increase. Several features of copy number control that are shown to be crucial for the understanding of ColE1-type plasmids still remain to be experimentally characterized. It is shown how steady-state properties reflect dynamics at the heart of regulation and therefore can be used to discriminate between fundamentally different copy number control mechanisms. The experimental tests of the predictions made require carefully planned assays, and some suggestions for suitable experiments arise naturally from the present work. It is also discussed how the presence of the Rom protein may affect dynamic qualities of copy number control.

Bacteriocin Plasmids↗
PubMed1997

Release factor RF3 abolishes competition between release factor RF1 and ribosome recycling factor (RRF) for a ribosome binding site.

The dependence of the rate of ribosomal recycling (from initiation via protein elongation and termination, and then back to initiation) on the concentrations of release factor RF1 and the ribosome recycling factor (RRF) has been studied in vitro. High RF1 concentration was found to reduce the rate of ribosomal recycling and the extent of this reduction depended on stop codon context. The inhibitory effect of high RF1 concentrations can be reversed by a corresponding increase in RRF concentration. This indicates that RF1 and RRF have mutually exclusive and perhaps overlapping binding sites on the ribosome. Addition of release factor RF3 to the translation system abolishes the inhibitory effect of high RF1 concentration and increases the overall rate of ribosome recycling. These data can be explained by a three-step model for termination where the first step is RF1-promoted hydrolysis of peptidyl-tRNA. The second step is an intrinsically slow dissociation of RF1 which is accelerated by RF3. The third step, catalysed by RRF and elongation factor G, leads to mobility of the ribosome on mRNA allowing it to enter a further round of translation. In the absence of RF3, RF1 can re-associate rapidly with the ribosome after peptidyl-tRNA hydrolysis, preventing RRF from entering the ribosomal A-site and thereby inhibiting ribosomal recycling. The overproduction of RF1 in cells deficient in RRF or lacking RF3 has effects on growth rate predicted by the in vitro experiments.

Bacterial Proteins↗
PubMed1997

An A to U transversion at position 1067 of 23 S rRNA from Escherichia coli impairs EF-Tu and EF-G function.

Escherichia coli ribosomes with an A to U transversion at nucleotide 1067 of their 23 S rRNA are impaired in their effective association rate constants (kcat/KM) for both EF-Tu and EF-G binding. In addition, the times that EF-G and EF-Tu spend on the ribosome during elongation are significantly increased by the A to U transversion. The U1067 mutation impairs EF-Tu function more than EF-G function. The increase in the time that EF-Tu remains bound to ribosome is caused, both by a slower rate of GTP-hydrolysis in ternary complex and by a slower EF-Tu.GDP release from the mutated ribosomes. There is, at the same time, no change in ribosomal accuracy for aminoacyl-tRNA recognition. With support from these new data we propose that nucleotide 1067 is part of the ribosomal A-site where it directly interacts with both EF-G and EF-Tu.

Adenine↗
PubMed1997

Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner.

Ribosomes complexed with synthetic mRNA and peptidyl-tRNA, ready for peptide release, were purified by gel filtration and used to study the function of release factor RF3 and guanine nucleotides in the termination of protein synthesis. The peptide-releasing activity of RF1 and RF2 in limiting concentrations was stimulated by the addition of RF3 and GTP, stimulated, though to a lesser extent, by RF3 and a non-hydrolysable GTP analogue, and inhibited by RF3 and GDP or RF3 without guanine nucleotide. With short incubation times allowing only a single catalytic cycle of RF1 or RF2, peptide release activity was independent of RF3 and guanine nucleotide. RF3 hydrolysis of GTP to GDP + P(i) was dependent only on ribosomes and not on RF1 or RF2. RF3 affected neither the rate of association of RF1 and RF2 with the ribosome nor the catalytic rate of peptide release. A model is proposed which explains how RF3 recycles RF1 and RF2 by displacing the factors from the ribosome after the release of peptide.

Bacterial Proteins↗